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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

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Published on: April 11, 2014

Measurement of ascorbic acid and dehydroascorbic acid in biological samples.

Jens Lykkesfeldt¹

¹Royal Veterinary and Agricultural University, Copenhagen, Denmark.

Current Protocols in Toxicology

|October 19, 2010

Summary

Accurate measurement of ascorbic acid and dehydroascorbic acid, key oxidative stress biomarkers, is challenging. This study details a reversed-phase HPLC method using uric acid as an internal standard to improve dehydroascorbic acid determination accuracy.

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Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Published on: May 12, 2020

Area of Science:

Biochemistry
Analytical Chemistry
Biomarker Analysis

Background:

Ascorbic acid and dehydroascorbic acid are crucial biomarkers for oxidative stress.
Accurate measurement of these labile compounds presents significant challenges in sample handling and analysis.
Current dehydroascorbic acid determination often relies on indirect subtraction methods, demanding precise quantification.

Purpose of the Study:

To present a detailed method for ascorbate analysis in biological samples.
To address common problems and pitfalls in measuring ascorbic acid and dehydroascorbic acid.
To introduce an improved analytical approach for accurate dehydroascorbic acid quantification.

Main Methods:

Utilized reversed-phase High-Performance Liquid Chromatography (HPLC) with colorimetric detection.
Incorporated co-analysis of isoascorbic acid and uric acid.
Employed uric acid as an endogenous intrasample standard to enhance accuracy.

Main Results:

The described HPLC method allows for accurate quantification of both ascorbic acid and total ascorbic acid.
The use of uric acid as an internal standard significantly improves the accuracy of dehydroascorbic acid calculation.
Identified and discussed common challenges in sample collection and analytical procedures.

Conclusions:

Accurate determination of dehydroascorbic acid is achievable with meticulous sample handling and quantitative reduction.
The proposed HPLC method with uric acid as an internal standard offers a robust approach for oxidative stress biomarker analysis.
This method enhances the reliability of dehydroascorbic acid measurements in biological samples.